Antimicrobial susceptibility testing (AST) is essential in clinical microbiology for selecting appropriate antibiotic therapy and monitoring resistance trends. It determines whether a bacterial isolate is susceptible, intermediate, or resistant to specific antimicrobial agents.
Disk Diffusion (Kirby-Bauer) Method
Filter paper disks impregnated with standardized concentrations of antibiotics are placed on an agar plate inoculated with the test organism at a defined turbidity (0.5 McFarland standard). After 16-24 hours of incubation, zones of inhibition are measured in millimeters and compared to CLSI or EUCAST breakpoint tables. The result is reported as Susceptible (S), Intermediate (I), or Resistant (R), with zone size correlating inversely with minimum inhibitory concentration (MIC). This method is simple, inexpensive, and flexible (any antibiotic disk can be tested), but it is not suitable for slow-growing or fastidious organisms and is qualitative rather than quantitative.
Broth Dilution Methods
Broth microdilution is performed in 96-well microtiter plates with serial twofold dilutions of antibiotics in Mueller-Hinton broth. Each well is inoculated with approximately 5x10^5 CFU/mL, and the MIC is the lowest antibiotic concentration that inhibits visible bacterial growth after 16-20 hours of incubation. Broth macrodilution uses the same principle in test tubes and is suitable for smaller numbers of isolates. MIC values provide quantitative data useful for pharmacokinetic/pharmacodynamic (PK/PD) analysis and detection of low-level resistance.
Agar Dilution
Agar plates containing serial dilutions of antibiotics are inoculated with a standardized bacterial suspension using a replicator (Steers or Denley multipoint inoculator). The MIC is read as the lowest concentration that completely inhibits growth, and multiple isolates (up to 36) can be tested simultaneously on a single plate. This method is considered the reference standard for MIC determination, particularly for fastidious organisms and anaerobes.
Gradient Diffusion (Etest)
A plastic strip with a predefined continuous antibiotic gradient on one side and an MIC reading scale on the other is placed on an inoculated agar plate. After incubation, an elliptical zone of inhibition intersects the strip at the MIC value, read directly from the scale. This method combines the convenience of disk diffusion with quantitative MIC data and is useful for single isolates or when broth dilution is impractical.
Automated Systems
Vitek 2 (bioMerieux), BD Phoenix, and MicroScan WalkAway automate AST using miniaturized broth microdilution in proprietary panels with growth detection by turbidity or fluorescence. Results are typically available in 4-12 hours, significantly faster than manual methods. Automated systems include integrated expert rules for detecting unusual resistance patterns and suggesting corrective actions.
Specialized Testing
Detection of extended-spectrum beta-lactamase (ESBL) uses a combination disk test with cefotaxime and ceftazidime with and without clavulanic acid; a ≥5 mm increase in zone diameter confirms ESBL production. Carbapenemase detection is performed using the modified carbapenem inactivation method (mCIM) and EDTA-modified CIM (eCIM) to distinguish serine- and metallo-carbapenemases. Inducible clindamycin resistance (D-test) involves placing erythromycin and clindamycin disks 15-20 mm apart; flattening of the clindamycin zone (D-shaped) indicates inducible macrolide-lincosamide-streptogramin B (MLSB) resistance. Vancomycin MIC determination by broth microdilution or Etest is preferred for detecting vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA).
Interpretation and Clinical Relevance
Clinical breakpoints are established by CLSI (Clinical and Laboratory Standards Institute) and EUCAST (European Committee on Antimicrobial Susceptibility Testing) based on MIC distributions, PK/PD data, and clinical outcomes. A result of Susceptible (S) indicates a high likelihood of therapeutic success with standard dosing; Resistant (R) indicates a high likelihood of therapeutic failure; and Intermediate (I) indicates uncertain efficacy, potentially requiring higher doses or alternative routes. AST results guide targeted therapy, reducing broad-spectrum antibiotic use and slowing resistance development. Cumulative AST data (antibiograms) are used for local epidemiology, formulary decisions, and infection control.
